Pancreatic ductal adenocarcinoma (PDAC) is the most deadly common cancer with no early detection methods or effective treatments. Understanding the pathological progression and molecular mechanisms of this disease is crucial to develop new methods for early diagnosis and treatment of PDAC. No effective drugs are developed to target the most frequently mutated four genes in PDAC, KRAS, CDKN2A/p16, TP53 and SMAD4. Thus, identifying other PDAC driver genes is crucial for developing therapeutic targets and studying drug resistance. We have established a platform to sort and culture normal pancreatic acinar and ductal cells from human donor and demonstrated that we are able to engineer these cells with oncogenic KRAS and inactivation of p16, TP53, and SMAD4 to generate invasive PDAC in a xenograft mouse model. Our previous data showed that oncogenic KRAS alone is not able to generate tumors, suggesting that additional driver mutation(s) are required for tumorigenesis. In this proposed project, we will employ our unprecedented model as well as a gene editing technique to screen ~200 potential tumor suppressor driver genes in PDAC to identify the driver mutations that contribute to the initiation and progression of PDAC. We will use these genetically engineered human cells to investigate how the driver mutations could affect drug sensitivity in these cells. To achieve our research goals, two specific aims are proposed: Aim 1. to Identify tumor suppressor genes that can promote acinar cells to tumors; Aim 2. to assess the therapeutic profile of the engineered acinar and ductal derived cells. Our model of using genetically engineered normal human acinar cells represents one of the best models to recapitulate the progression of PDAC in human cells, and thus will lead to successful identification of PDAC driver mutations, a key step towards better understanding tumor biology and development of targeted therapies, especially for precision oncology. Our overall goal is to elucidate the molecular mechanisms which may lead to the development of new diagnostic and treatment methods for PDAC that can increase survival and improve patient outcomes.